Stabilization of eukaryotic ribosomal termination complexes by deacylated tRNA

Susorov, Denis; Mikhailova, Tatiana; Иванов, А. В.; Sokolova, Elizaveta; Alkalaeva, Elena

doi:10.1093/nar/gkv171

Cited by 17 publications

(18 citation statements)

References 51 publications

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“…In particular, it has been shown that deacylated tRNA in the E-site stabilises the unrotated conformation of the PRE complex, with the A-and P-tRNAs in the classical states (5). Furthermore, we also have previously shown the same mechanism of stabilisation regarding eukaryotic termination complexes, in which deacylated tRNA in the P-site was produced by peptidyl-tRNA hydrolysis (43).…”

Section: Condition Requirements For Reverse Translocationmentioning

confidence: 54%

“…The 40S and 60S ribosomal subunits, as well as rabbit translation factors eIF2, eIF3, eEF1H, and eEF2, were purified from a rabbit reticulocyte lysate as described (30,43). The human translation factors eIF1, eIF1A, eIF4A, eIF4B, ∆eIF4G, ∆eIF5B, and eIF5 were produced as recombinant proteins in Escherichia coli strain BL21 with subsequent protein purification on Ni-NTA agarose and ion-exchange chromatography (30,47).…”

Section: Ribosomal Subunits and Translation Factorsmentioning

confidence: 99%

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Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

Susorov

Zakharov

Shuvalova

et al. 2018

Journal of Biological Chemistry

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During protein synthesis, a ribosome moves along the mRNA template and, using aminoacyl-tRNAs, decodes the template nucleotide triplets to assemble a protein amino acid sequence. This movement is accompanied by shifting of mRNA-tRNA complexes within the ribosome in a process called translocation. In living cells, this process proceeds in a unidirectional manner, bringing the ribosome to the 3′-end of mRNA, and is catalyzed by the GTPase translation elongation factor 2 (EF-G in prokaryotes, eEF2 in eukaryotes). Interestingly, the possibility of spontaneous backward translocation has been shown in vitro for bacterial ribosomes, suggesting a potential reversibility of this reaction. However, this possibility has not yet been tested in eukaryotic cells. Here, using a reconstituted mammalian translation system, we show that eukaryotic elongation factor eEF2 catalyzes ribosomal reverse translocation at one mRNA triplet. We found that this process requires a cognate tRNA in the ribosomal E-site and cannot occur spontaneously without eEF2. The efficiency of this reaction depended on the concentrations of eEF2 and cognate tRNAs and increased in the presence of nonhydrolyzable GTP analogues. Of note, ADP-ribosylation of eEF2 domain IV blocked reverse translocation, suggesting a crucial role of interactions of this domain with the ribosome for the catalysis of the reaction. In summary, our findings indicate that eEF2 is able to induce ribosomal translocation in forward and backward directions highlighting the universal mechanism of tRNA-mRNA movements within the ribosome.

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Section: Condition Requirements For Reverse Translocationmentioning

confidence: 54%

Section: Ribosomal Subunits and Translation Factorsmentioning

confidence: 99%

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

Susorov

Zakharov

Shuvalova

et al. 2018

Journal of Biological Chemistry

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“…Pre-termination complexes (preTCs) were assembled in vitro as described ( 36 ) and used in peptide release assays and conformational rearrangement analyses by toe-print assays ( 37 ). For peptide release assays aliquots containing 0.2 pmol of the preTCs were incubated at 37°C for 3 min with 0.6 pmol of eRF1/3 and 5 pmol of PABP.…”

Section: Methodsmentioning

confidence: 99%

PABP enhances release factor recruitment and stop codon recognition during translation termination

et al. 2016

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Poly(A)-binding protein (PABP) is a major component of the messenger RNA–protein complex. PABP is able to bind the poly(A) tail of mRNA, as well as translation initiation factor 4G and eukaryotic release factor 3a (eRF3a). PABP has been found to stimulate translation initiation and to inhibit nonsense-mediated mRNA decay. Using a reconstituted mammalian in vitro translation system, we show that PABP directly stimulates translation termination. PABP increases the efficiency of translation termination by recruitment of eRF3a and eRF1 to the ribosome. PABP's function in translation termination depends on its C-terminal domain and its interaction with the N-terminus of eRF3a. Interestingly, we discover that full-length eRF3a exerts a different mode of function compared to its truncated form eRF3c, which lacks the N-terminal domain. Pre-association of eRF3a, but not of eRF3c, with pre-termination complexes (preTCs) significantly increases the efficiency of peptidyl–tRNA hydrolysis by eRF1. This implicates new, additional interactions of full-length eRF3a with the ribosomal preTC. Based on our findings, we suggest that PABP enhances the productive binding of the eRF1–eRF3 complex to the ribosome, via interactions with the N-terminal domain of eRF3a which itself has an active role in translation termination.

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“…Our metagene plots lack the pronounced peak at the stop codon, both in the case of wild type and new1Δ datasets. This is not unexpected as ribosomal post-termination complexes are more sensitive to ionic strength than elongating ribosomes (58) and require stabilisation by cycloheximide that was specifically omitted in our Ribo-Seq protocol (61).…”

Section: New1 Depletion Leads To Ribosome Queuing Upstream Of 3'-termmentioning

confidence: 78%

A role for theSaccharomyces cerevisiaeABCF protein New1 during translation termination

Kasari

Pochopien

Margus

et al. 2019

Preprint

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Translation on the ribosome is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related ABCF ATPase, New1, is encoded by a non-essential gene with a cold sensitivity and ribosome assembly defect knock-out phenotype. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide ribosome assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed a combination of yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3.The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 associated ATPases belonging to the ATP-binding cassette (ABC) type F (ABCF) protein family.Eukaryotic representatives of this protein family include initiation factor ABCF1/ABC50 (7,8), elongation factor eEF3 (9,10) as well as Gcn20 -a key component of general amino acid control pathway (11).Elongation factor eEF3 is the most well-studied eukaryotic ribosome-associated ABCF ATPase.While early analyses of the evolutionary distribution of eEF3 concluded that it is a fungi-specific translational factor (12) eEF3-like homologues have more recently been found in non-fungal species, such as oomycete Phytophthora infestans (13), choanoflagellates and various algae (14). eEF3 is essential both for viability of the yeast Saccharomyces cerevisiae (10) and for peptide elongation in a reconstituted yeast translational system (15,16). While biochemical experiments suggest a secondary function for eEF3 in ribosome recycling (17), ribosome profiling analysis of eEF3-depleted S. cerevisiae suggests that translation elongation is the primary function of eEF3 in the cell (18). A cryo-electron microscopy (cryo-EM) reconstruction of eEF3 on the ribosome localizes the factor within the vicinity of the ribosomal E-site (9), providing a structural explanation for the biochemical observations of E-site tRNA displacement from the ribosome in the presence of eEF3 and ATP (19).The S. cerevisiae genome encodes 29 ABC ATPases, of which five belong to the ABCF subfamily (20). Of these, eEF3, Hef3/eEF3B and New1 together form a group with a distinctive subdomain architecture relative to the other ABCFs (14). Hef3/eEF3B is the closest homologue of eEF3 -84% identical on the amino acid level (21) -and is, most likely, a second copy of eEF3 originating from whole-genome duplication in the Saccharomyces lineage (22) (Figure 1A). The second closest homologue is New1 (20) -encoded by a non-essential gene with a cold sensitivity knock-out phenotype (23) (Figure 1A)...

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Stabilization of eukaryotic ribosomal termination complexes by deacylated tRNA

Cited by 17 publications

References 51 publications

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

PABP enhances release factor recruitment and stop codon recognition during translation termination

A role for theSaccharomyces cerevisiaeABCF protein New1 during translation termination

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